Concentraction of amylolytic enzymes



Patented Sept. 8, 1953 CONCENTRATION OF AMYLOLYTIC ENZYMES Kenneth J. Goering, Bozeman, Mont., and Victor 0. Bruski, Linden, N. J assignors to Research Corporation, New York, N. Y., a corporation of New York No Drawing. Application September 5, 1951, Serial No. 245,271

8 Claims. (Cl. 195-66) This invention relates to the concentration of amylolytic enzymes produced by submerged cultures of various fungi.

We have discovered that by suitable adjustment of temperature and pH the amylolytic enzymes produced by fungi can be completely adsorbed on as little as A of the mycelium present in the growth medium. By elution from this mycelium a concentration of 20-40 fold can be obtained and at the same time the enzyme is separated from the contaminating carbohydrates, salts, and from over 80% of the protein present. A second adsorption will allow an additional 2-3 fold concentration.

This procedure provides a very inexpensive method of concentration, eliminating the usual methods of concentration under reduced pressure, and precipitation by alcohol or other organic solvents. Furthermore a much purer material is obtained.

According to the present invention the culture is grown under the usual conditions of forced aeration on a starch and nitrogen containing medium. When opimum amylolytic activity is obtained the culture is well mixed, about onefifth of the total volume of fermentation liquor including a proportionate amount of mycelium is transferred to a large tank, and the balance of the mycelium is removed from the remaining fermentation liquor by screening or filtration after first adjusting the pH to 8 with NaOH. This filtrate is added to the original volume withdrawn containing the mycelium, the pH adjusted to 5.0 with glacial acetic acid, the temperature adjusted to 37 C. and the entire mixture gently agitated for hour. At the end of this time the mycelium is removed by filtration and the filtrate discarded. After washing twice with water adjusted to pH 5-6, the mycelium is resuspended in a minimum of water /2o /4o original volumeof the fermentation) to which has been added 0.27 pound of crystalline boric acid 0.62 pound of potassium chloride and 8.0 pounds of sodium chloride per 100 gallons of liquor. The pH is then adjusted to 8.5 with NaOH with careful stirring. After hour the mycelium is removed from the extracted enzyme by filtration and a concentration of twenty to forty fold has been reached.

This process can be repeated using a concentrated amylolytic enzyme allowing a concentration of 50-120 times that in the original fermentation. The concentrated enzyme may then be sprayed on dry starch, dextrin or sugar 7 potency of present commercial products eliminating the costly steps of evaporation, precipitation with alcohol or other organic solvents, vacuum drying and grinding. This not only results in savings in both cost of operation and equipment but the over-all recoveries are better since losses are encountered in all of the steps mentioned above.

If an-even more concentrated liquor is desired the material from the second adsorption can be concentrated by vacuum distillation with no loss of activity. Since at this stage the total volume handled is only 1/100 ofthe original, the size of the equipment in the final step can be very small compared to the rest of the operation. With this third step concentrations of 400-600 times are readily obtained.

The principles of the invention are further illustrated by the following example:

in the proportions indicated is advantageously used:

Corn starch per cent by weight 1.5 KNOs d0 0A CEtCOa do 0.1 KHzPOi do 0.025 MgSOi do .01 KCl do .01 FeSO4-7H2O p. p. m .44 Zl'lSO4-7H2O p. p. m 1. CUSO4'5H2O p. p. m.-- 0.22

10 liters of this composition is double sterilized on successive days and inoculated with a pure culture of Aspergillus oryzae. The medium. is maintained at a temperature of 32-34 C. and aerated at a rate of volume of air per volume of medium per minute. After 72-96 hours the alpha amylase activity'reaches a value of'from 15-40 units per ml. a

After the 10 liters of fermentation liquor has reached its optimum amylase activity its pH is checked and adjusted to 8 if necessary and the mycelium removed by filtration. The mycelium is discarded except about 20% of total g. containing moisture). This is added to the filtrate andthe pH adjusted to 5 with glacial acetic acid (10 ml). Themixture'is held at 37 C. with gentle stirring for hour and the mycelium removed by filtration. The filtrate has an activity of 4.0.units/ml. comparedto 15for the originalfermentation. j

The mycelium is then placed in 50 ml. of borate 3 buiier pH 8.5, 1 g. of NaCl added and the volume made up to 500 ml. Borate buffer contains 50 ml. 0.2M K01, 50 ml. 0.2M H3303, and 8.5 ml. 0.2M NaOH, diluted to .200 m1. This mixture is gently stirred for one hour: and the mycelium removed by filtration. The filtrate has an activity of 240 units per ml.

Protein removed 85% :Recoveries' as high as 90% and concentrations ,of-over :35 times have been obtained.

; In:;;genera1, thepptimum temperature for the .yconcentrationi step is from about-30 to about 40 0., and the optimum pH is aboutr5. The method is applicable togtheconeentration of amylolytic-enzymesproduced by the submerged culture of other'fungi such-as Aspergillus niger.

Concentrated enzyme solutions can be concentrated by 51168941119: procedure as indicated in the following table:

1 i Total Total alpha K eldahl volume! amylase, N, mg./ g flggg :ml. units/ml. -ml. (ing) Orig. culture liquor 206100 15 .1475 2,950 1st elute 550 430 .855 470 2d elute 200 1. 000 l. 80 360 It will be seen that a concentration of 73 times the original is obtained and that approximately 87% of the Kjeldal'il nitrogen is eliminated in two adsorption and elutions.

The'followmgexampleshows-thesuitability of "further "c'oncemirating the concentrate from the 'fo'regom'g tableby vacuumdistillation:

90 mlJdf'the l'l'otlunitmaterial"is' concentratedby vacuum' di'stillatiomtoia ml. (a little "more than i 6 times) "The concentrate has "an 'activitypf 75000 units'perml. "Recoveryis-100% :withintthezlimits of experimental error on the :measuring'ivolumesxof these concentrated solutions.

:Ihis procedure has theadvantage that when -.the J final evaporation step is carried out very small equipment canbe used and the cost is low because the solution has 'already been concentrated 100 times.

We claim: 7

LA method of concentrating alpha amylase producedbythesubmerged culture of fungi which comprises-contacting the liquid portion of the culture medium with a minor portion of the mycelium of the fungus at a pH or abut'5 whereby to adsorb alphaamylase from the liquor upon the mycelium, separatin'g' the mycelium from the liquid, and'extracting the adsorbed alpha am- .ylasei-ro'm the'imycelium with an aqueousmedium at a pet of .about815.

2. A method of concentrating alpha amylase produced lay-the submergedculture of fungi which comprises contacting the liquid portion of the 4 culture medium with about 20% of the myceliuni of the fungus from the culture medium at a pH of about 5 whereby to adsorb alpha amylase from the liquor-upon the mycelium, separating the mycelium from the liqu-idwarid ei'ztracting the enzyme adsorbed alpha amylase from the myce- .lium with an aqueous medium at a pH of about 815.

13..A :method \of concentrating alpha amylase from the submerged culture of Aspergz'llus org ace,

by to adsorb-alphaiamylase from the liquor upon the mycelium, separating the mycelium from the r liquid. and" extracting the adsorbed alpha amylase from'the mycelium with an aqueous medium at a-pH of about 8.5.

4. A method of concentrating alpha amylase fromthe'submerged culture of .Aspergillus oryzae, which comprises. contacting the'liquid; portion of the culture medium with about'T20'% of'the' mycelium of the fungus from'the culture medium at a pH of about5 whereby to adsorb alpha amylasefrom the liquor upon'the'myceliumfsep arating' the mycelim'n from the liquid andeX- tracting the adsorbed alphaamylasefromthe mycelium with an 'aqueous"me"dium'at-apH -of about"8.'5.

5. A method of concentrating 'Jalpha "amylase from the submerged eulture'of Aspev'giil'us orgzae, which comprises contacting the liquid portion-of the culture mediurrr with a, minorportion-of the myeelium of the fungus at "a err-oi "about "5 whereby to adsorb alpha amylasefrom the liquor upon the myce'liumandata; temperature offrom about '36 20 0.; separatingthemycelium from the liquid and extracting the "adsorbed "alpha amylase fromthemyceliumwvithanaqueous medium at a pH of about 8.5.

"6. A method of "concentrating alpha-amylase from the submergedculture of'AspergiZZusorg/eae, which comprises contacting"the liquiwportionof the culture 'medium"with" a minor portion *of" the myceiimn of the fungus at a "pH'of about "5 whereby to adsorb alpha amylasefifrom' theliquor upon'the "mycelium, separating the myeelium fromthe liquid" and-extracting theadso'rbe'd alpha amylase fronrthe myeelium with-an aoueous sodium chloride solution -at-'a pH'of about-825.

"7. The method definedin-"claim-1w-hereinthe enzyme is subjected to at leastone further -aw sorption on -a""m-inor' portion-of=myclium ata p-H of aboutfi-andextractio'n from the mycelium at a pI-Iofabout-Sfi.

8. The method defined in claim l wherein the enzyme is subj ected --to at -least-v-one further =adsorption on a minor portion-of mycelium atsa pH of about 5- and extraction irom the mycelium at a pH of about 8. 5--and--is thereafter further concentrated-by vacuum evaporation.

..KENNETH .J. GOERING.

VICTOR-.QZBRHSKI.

.Referenees Cited in the "file of 1 thisypatent UNITED -STAT-E-S PATE-NTS Number Name Date 1,263,817 Takamine 'Apr "23, 1918 1,564,385 Waksnian lDec. 8, I925 OTHERiL-REEERENCES Caldwell et'al; "J. "A: C. 'Sc-59 (l'937'),-pages "Caldwell" et'aL: '27 Biol. fithem. 1'6l"(1945). 

1. A METHOD OF CONCENTRATING ALPHA AMYLASE PRODUCED BY THE SUBMERGED CULTURE OF FUNGI WHICH COMPRISES CONTACTING THE LIQUID PORTION OF THE CULTURE MEDIUM WITH A MINOR PORTION OF THE MY CELIUM OF THE FUNGUS AT A PH OF ABOUT 5 WHEREBY TO ADSORB ALPHA AMYLASE FROM THE LIQUOR UPON THE MYCELLIUM, SEPARATING THE MYCELIUM FROM THE LIQUID, AND EXTRACTING THE ADSORBED ALPHA AMYLASE FROM THE MYCELIUM WITH AN AQUEOUS MEDIUM AT A PH OF ABOUT 8.5. 